lab meeting 2013.05.13
Post on 15-Jan-2016
29 Views
Preview:
DESCRIPTION
TRANSCRIPT
Lab meeting 2013.05.13
Nguyen Thi Dai Trang
Electroporation of K562, Hela, IM9
Protocol1. 2x106 cells
2. PBS wash 2 times
3. Suspend in 90µl PBS
4. Add 10µl linear vector (10µg). Total volume now is 100µl
5. Incubate on ice 10 min
6. Electroporation 3µF, 800 V
7. Spread on 100 mm disk with full RPMI
8. 48 hours, add Puromycin to selection
U2AF1
RESULTS
• All K562, Hela, IM9 cells die
• Assumed reasons:
• + capacitance, voltage, cell density (3µF, 800 V, 2x106)
+ kit for purification DNA enclosed toxins for mamalian cells
U2AF1
Source: http://www.biorad-ads.com/transfection_protocols/index.html?source_wt=transfectionprotocols_surl
Solution
Try 1 more time using following condition:
• For Hela: 166V, 500µF, 1x107
Step 3: cDNA SRSF2 digested with NheI, XhoI
Source: http://tools.neb.com/NEBcutter2/showdig.php?name=e051c50b-SRSF2-cloning_seq_C-
cDNA of SRSF2 gene was composed of 231 aa and 714 bp.
XhoI C’TCGA,G
NheI G’CTAG,C
Enzyme Specificity
SRSF2
Step 3: Restriction Endonuclease NheI, XhoI
Restriction Digestion for Ligation Reaction
NheI 1 µl
XhoI 1 µl
10X NEBuffer (1X) 5 µl
BSA 1 µl
cDNA SRSF2 (40-46 ng/ µl) 5 µl
Distilled water 37 µl
Total: 50 µl
• Incubation time: overnight
• Incubation temp: 37 °C
After digested by RE NheI & XhoI
Digested cDNA U2AF1
(546 bp)
K562 Hela IM9
708 bp
Ready for ligation into pcDNA3.1
SRSF2
Step 3: pcDNA3.1 digested with NheI, XhoI
Source: http://tools.neb.com/NEBcutter2/showdig.php?name=a4b78c98-pcDNA_3.1_seq
XhoI C’TCGA,G
NheI G’CTAG,C
Enzyme Specificity
SRSF2
Step 3: Restriction Endonuclease NheI, XhoI
Ready for ligation with cDNA SRSF2
1kb plus DNA ladder
5000 bp
Digested pcDNA 3.1(5338bp)
Restriction Digestion for Ligation Reaction
NheI 1 µl
XhoI 1 µl
10X NEBuffer (1X) 5 µl
BSA 1 µl
pcDNA3.1 (55 ng/ µl) 1 µl
Distilled water 41 µl
Total: 50 µl
• Incubation time: overnight
• Incubation temp: 37 °C
SRSF2
Step 4: Ligate into expression vector pcDNA3.1
Ligation Reaction
2X Rapid Ligation buffer, T4 DNA ligase 5 µl
pcDNA3.1 vector (55 ng/ µl) 1 µl
cDNA U2AF1 (55 ng/ µl) 3 µl
T4 DNA ligase (3 units/µl) 1 µl
Distilled water 10 µl
Total: 20 µl
• Incubation time: overnight
• Incubation temp: 4°C
SRSF2
This week’s plan
• Ligate the insert into the pcDNA3.1 vector and transform into E. coli.
• Select transformants on LB plates containing 100 μg/ml ampicillin.
• Analyze and select transformants for the presence of insert by PCR, restriction digestion and sequencing.
• Try electroporation again with Hela
SRSF2
U2AF1
top related